Editorials; complex trait; congenic strain; quantitative trait loci; Raet1e
Purpose of review
The paraoxonase (PON) gene family includes 3 members, PON1, PON2, and PON3. In vitro and mouse studies have demonstrated that all three PONs are athero-protective. Some but not all human epidemiologic studies have observed associations between PON gene polymorphisms and risk of cardiovascular disease (CVD). In this review, we summarize studies published within the past year elucidating involvement of PON1 and PON2 in oxidative stress, cardiovascular disease, and innate immune responses.
In a prospective study, the PON1 192QQ genotype and low PON1 activity were associated with increased systemic oxidative stress and increased risk for cardiovascular disease. PON1 expression protected against Pseudomonas aeruginosa lethality in Drosophila, suggesting that PON1 can interfere with quorum sensing in vivo. PON2 attenuated macrophage triglyceride accumulation via inhibition of diacylglycerol acyltransferase 1. Over-expression of PON2 protected against endoplasmic reticulum (ER) stress-induced apoptosis when the stress was induced by interference with protein modification but not when ER stress was induced by Ca ++ deregulation.
Both mouse and human studies have demonstrated the anti-oxidative and athero-protective effects of PON1. The mechanisms by which PON2 exerts its athero-protective effects are emerging. Large-scale epidemiologic studies are needed to further examine the relationship between PON2 genetic polymorphisms and risk for CVD. Elucidation of the physiological substrates of the PON proteins is of particular importance to further advance this field.
Atherosclerosis; high density lipoprotein; paraoxonases; oxidative stress; quorum sensing
Genetic variations in blood cell parameters can impact clinical traits. We report here the mapping of blood cell traits in a panel of 100 inbred strains of mice of the Hybrid Mouse Diversity Panel (HMDP) using genome-wide association (GWA). We replicated a locus previously identified using linkage analysis in several genetic crosses for mean corpuscular volume 1 and a number of other red blood cell traits on distal chromosome 7. Our peak for SNP association to MCV occurred in a linkage disequilibrium (LD) block spanning from 109.38 to 111.75 Mb that includes Hbb-b1, the likely causal gene. Altogether, we identified 5 loci controlling red blood cell traits (on chromosomes 1, 7, 11, 12, and 16), and four of these correspond to loci for red blood cell traits reported in a recent human GWA study. For white blood cells, including granulocytes, monocytes, and lymphocytes, a total of six significant loci were identified on chromosomes 1, 6, 8, 11, 12 and 15. An average of ten candidate genes were found at each locus and those were prioritized by examining functional variants in the HMDP such as missense and expression variants. These new results provide intermediate phenotypes and candidate loci for genetic studies of atherosclerosis and cancer as well as inflammatory and immune disorders in mice.
blood cell traits; genetics; association; linkage; red cell; white cell; mice
Recent evidence suggests that dendritic cells may play an important role in atherosclerosis. Based primarily on previous in vitro studies, we hypothesized that granulocyte macrophage colony-stimulating factor (GM-CSF)-deficient mice would have decreased dendritic cells in lesions.
Methods and Results
To test this, we characterized gene targeted GM-CSF−/− mice crossed to hypercholesterolemic low-density lipoprotein receptor null mice. Our results provide conclusive evidence that GM-CSF is a major regulator of dendritic cell formation in vivo. Aortic lesion sections in GM-CSF−/− low-density lipoprotein receptor null animals showed a dramatic 60% decrease in the content of dendritic cells as judged by CD11c staining but no change in the overall content of monocyte-derived cells. The GM-CSF–deficient mice exhibited a significant 20% to 50% decrease in the size of aortic lesions, depending on the location of the lesions. Other prominent changes in GM-CSF−/− mice were decreased lesional T cell content, decreased autoantibodies to oxidized lipids, and striking disruptions of the elastin fibers adjacent to the lesion.
Given that GM-CSF is dramatically induced by oxidized lipids in endothelial cells, our data suggest that GM-CSF serves to regulate dendritic cell formation in lesions and that this, in turn, influences inflammation, plaque growth and possibly plaque stability.
atherosclerosis; dendritic cells; elastin; GM-CSF; macrophages
To determine the efficacy of long-term anti-miR-33 therapy on the progression of atherosclerosis in high fat, high cholesterol-fed Ldlr−/− mice.
Methods and Results
Ldlr−/− mice received saline, or control or anti-miR-33 oligonucleotides once a week for 14 weeks. The treatment was effective, as measured by reduced levels of hepatic miR-33 and increased hepatic expression of miR-33 targets. Analysis of plasma samples revealed an initial elevation in HDL-cholesterol after two weeks of treatment that was not sustained by the end of the experiment. Additionally, we found a significant increase in circulating triglycerides in anti-miR-33-treated mice, compared to controls. Finally, examination of atheromata revealed no significant changes in the size or composition of lesions between the three groups.
Prolonged silencing of miR-33 fails to maintain elevated plasma HDL-cholesterol and does not prevent the progression of atherosclerosis in Ldlr−/− mice.
miR-33; atherosclerosis; HDL; VLDL; ABCA1
Genetic and gene expression studies have suggested an important role for KLF14 in metabolic disease. A new study now identifies a network of genes whose expression is associated with KLF14 variation in trans, providing a framework for understanding how KLF14 influences disease risk.
Systems genetics is an approach to understand the flow of biological information that underlies complex traits. It uses a range of experimental and statistical methods to quantitate and integrate intermediate phenotypes, such as transcript, protein or metabolite levels, in populations that vary for traits of interest. Systems genetics studies have provided the first global view of the molecular architecture of complex traits and are useful for the identification of genes, pathways and networks that underlie common human diseases. Given the urgent need to understand how the thousands of loci that have been identified in genome-wide association studies contribute to disease susceptibility, systems genetics is likely to become an increasingly important approach to understanding both biology and disease.
genetics; models, theoretical; pharmacogenetics; systems biology; atherosclerosis; heart failure; mapping
Identifying environmentally-specific genetic effects is a key challenge in understanding the structure of complex traits. Model organisms play a crucial role in the identification of such gene-by-environment interactions, as a result of the unique ability to observe genetically similar individuals across multiple distinct environments. Many model organism studies examine the same traits but under varying environmental conditions. For example, knock-out or diet-controlled studies are often used to examine cholesterol in mice. These studies, when examined in aggregate, provide an opportunity to identify genomic loci exhibiting environmentally-dependent effects. However, the straightforward application of traditional methodologies to aggregate separate studies suffers from several problems. First, environmental conditions are often variable and do not fit the standard univariate model for interactions. Additionally, applying a multivariate model results in increased degrees of freedom and low statistical power. In this paper, we jointly analyze multiple studies with varying environmental conditions using a meta-analytic approach based on a random effects model to identify loci involved in gene-by-environment interactions. Our approach is motivated by the observation that methods for discovering gene-by-environment interactions are closely related to random effects models for meta-analysis. We show that interactions can be interpreted as heterogeneity and can be detected without utilizing the traditional uni- or multi-variate approaches for discovery of gene-by-environment interactions. We apply our new method to combine 17 mouse studies containing in aggregate 4,965 distinct animals. We identify 26 significant loci involved in High-density lipoprotein (HDL) cholesterol, many of which are consistent with previous findings. Several of these loci show significant evidence of involvement in gene-by-environment interactions. An additional advantage of our meta-analysis approach is that our combined study has significantly higher power and improved resolution compared to any single study thus explaining the large number of loci discovered in the combined study.
Identifying gene-by-environment interactions is important for understand the architecture of a complex trait. Discovering gene-by-environment interaction requires the observation of the same phenotype in individuals under different environments. Model organism studies are often conducted under different environments. These studies provide an unprecedented opportunity for researchers to identify the gene-by-environment interactions. A difference in the effect size of a genetic variant between two studies conducted in different environments may suggest the presence of a gene-by-environment interaction. In this paper, we propose to employ a random-effect-based meta-analysis approach to identify gene-by-environment interaction, which assumes different or heterogeneous effect sizes between studies. Our approach is motivated by the observation that methods for discovering gene-by-environment interactions are closely related to random effects models for meta-analysis. We show that interactions can be interpreted as heterogeneity and can be detected without utilizing the traditional approaches for discovery of gene-by-environment interactions, which treats the gene-by-environment interactions as covariates in the analysis. We provide a intuitive way to visualize the results of the meta-analysis at a locus which allows us to obtain the biological insights of gene-by-environment interactions. We demonstrate our method by searching for gene-by-environment interactions by combining 17 mouse genetic studies totaling 4,965 distinct animals.
Circulating trimethylamine-N-oxide (TMAO) levels are strongly associated with atherosclerosis. We now examine genetic, dietary, and hormonal factors regulating TMAO levels. We demonstrate that two flavin monooxygenase family members, FMO1 and FMO3, oxidize trimethylamine (TMA), derived from gut flora metabolism of choline, to TMAO. Further, we show that FMO3 exhibits 10-fold higher specific activity than FMO1. FMO3 overexpression in mice significantly increases plasma TMAO levels while silencing FMO3 decreases TMAO levels. In both humans and mice, hepatic FMO3 expression is reduced in males compared to females. In mice, this reduction in FMO3 expression is due primarily to down-regulation by androgens. FMO3 expression is induced by dietary bile acids by a mechanism that involves the farnesoid X receptor (FXR), a bile acid-activated nuclear receptor. Analysis of natural genetic variation among inbred strains of mice indicates that FMO3 and TMAO are significantly correlated, and TMAO levels explains 11% of the variation in atherosclerosis.
To replicate the associations of leukocyte telomere length (LTL) with variants at four loci and to investigate their associations with coronary heart disease (CHD) and type II diabetes (T2D), in order to examine possible causal effects of telomere maintenance machinery on disease aetiology.
Four SNPs at three loci BICD1 (rs2630578 GγC), 18q12.2 (rs2162440 GγT), and OBFC1 (rs10786775 CγG, rs11591710 AγC) were genotyped in four studies comprised of 2353 subjects out of which 1148 had CHD and 566 T2D. Three SNPs (rs12696304 CγG, rs10936601G>T and rs16847897 GγC) at the TERC locus were genotyped in these four studies, in addition to an offspring study of 765 healthy students. For all samples, LTL had been measured using a real-time PCR-based method.
Only one SNP was associated with a significant effect on LTL, with the minor allele G of OBFC1 rs10786775 SNP being associated with longer LTL (β=0.029, P=0.04). No SNPs were significantly associated with CHD or T2D. For OBFC1 the haplotype carrying both rare alleles (rs10786775G and rs11591710C, haplotype frequency 0.089) was associated with lower CHD prevalence (OR: 0.77; 95% CI: 0.61–0.97; P= 0.03). The TERC haplotype GTC (rs12696304G, rs10936601T and rs16847897C, haplotype frequency 0.210) was associated with lower risk for both CHD (OR: 0.86; 95% CI: 0.75-0.99; P=0.04) and T2D (OR: 0.74; 95% CI: 0.61–0.91; P= 0.004), with no effect on LTL. Only the last association remained after adjusting for multiple testing.
Of reported associations, only that between the OBFC1 rs10786775 SNP and LTL was confirmed, although our study has a limited power to detect modest effects. A 2-SNP OBFC1 haplotype was associated with higher risk of CHD, and a 3-SNP TERC haplotype was associated with both higher risk of CHD and T2D. Further work is required to confirm these results and explore the mechanisms of these effects.
The lipin protein family of phosphatidate phosphatases has an established role in triacylglycerol synthesis and storage. Physiological roles for lipin-1 and lipin-2 have been identified, but the role of lipin-3 has remained mysterious. Using lipin single- and double-knockout models we identified a cooperative relationship between lipin-3 and lipin-1 that influences adipogenesis in vitro and adiposity in vivo. Furthermore, natural genetic variations in Lpin1 and Lpin3 expression levels across 100 mouse strains correlate with adiposity. Analysis of PAP activity in additional metabolic tissues from lipin single- and double-knockout mice also revealed roles for lipin-1 and lipin-3 in spleen, kidney, and liver, for lipin-1 alone in heart and skeletal muscle, and for lipin-1 and lipin-2 in lung and brain. Our findings establish that lipin-1 and lipin-3 cooperate in vivo to determine adipose tissue PAP activity and adiposity, and may have implications in understanding the protection of lipin-1-deficient humans from overt lipodystrophy.
Gene family; Knockout mouse; Adipogenesis; Triacylglycerol; Glycerolipid biosynthesis
Common cardiovascular diseases, such as atherosclerosis and congestive heart failure, are exceptionally complex, involving a multitude of environmental and genetic factors that often show nonlinear interactions as well as being highly dependent on sex, age, and even the maternal environment. Although focused, reductionistic approaches have led to progress in elucidating the pathophysiology of cardiovascular diseases, such approaches are poorly powered to address complex interactions. Over the past decade, technological advances have made it possible to interrogate biological systems on a global level, raising hopes that, in combination with computational approaches, it may be possible to more fully address the complexities of cardiovascular diseases. In this Review, we provide an overview of such systems-based approaches to cardiovascular disease and discuss their translational implications.
Many common diseases have an important inflammatory component mediated in part by macrophages. Here we used a systems genetics strategy to examine the role of common genetic variation in macrophage responses to inflammatory stimuli.
We examined genome-wide transcript levels in macrophages from 92 strains of the Hybrid Mouse Diversity Panel. We exposed macrophages to control media, bacterial lipopolysaccharide, or oxidized phospholipids. We performed association mapping under each condition and identified several thousand expression quantitative trait loci (eQTL), gene-by-environment interactions and several eQTL “hotspots” that specifically control LPS responses. We validated an eQTL hotspot in chromosome 8 using siRNA knock-down of candidate genes and identified the gene 2310061C15Rik, as a novel regulator of inflammatory responses in macrophages.
We have created a public database where the data presented here can be used as a resource for understanding many common inflammatory traits which are modeled in the mouse, and for the dissection of regulatory relationships between genes.
There is increasing clinical evidence that phospholipid oxidation products (Ox-PL) play a role in atherosclerosis. This review focuses on the mechanisms by which Ox-PL interact with endothelial cells, monocyte/macrophages, platelets, smooth muscle cells and HDL to promote atherogenesis. In the last few years major progress has been made in identifying these mechanisms. It has been recognized that Ox-PL promote phenotypic changes in these cell types that have long term consequences for the vessel wall. Individual Ox-PL responsible for specific cellular effects has been identified. A model of the configuration of bioactive truncated Ox-PL within membranes has been developed that demonstrates that the oxidized fatty acid moiety protrudes into the aqueous phase, rendering it accessible for receptor recognition. Receptors and signaling pathways for individual Ox-PL species are now determined and receptor independent signaling pathways identified. The effects of Ox-PL are mediated both by gene regulation and transcription independent processes. It has now become apparent that Ox-PL affects multiple genes and pathways some of which are pro-atherogenic and some are protective. However, at concentrations that are likely present in the vessel wall in atherosclerotic lesions, the effects promote atherogenesis. There have also been new insights on enzymes that metabolize Ox-PL and the significance of these enzymes for atherosclerosis. With the knowledge we now have on the regulation and effects of Ox-PL in different vascular cell types, it should be possible to design experiments to test the role of specific Ox-PL on the development of atherosclerosis.
Ox-PAPC; atherosclerosis; inflammation; phospholipids
Paraoxonase 3 (PON3) is a member of the PON family, which includes PON1, PON2, and PON3. Recently, PON3 was shown to prevent the oxidation of low-density lipoprotein in vitro. To test the role of PON3 in atherosclerosis and related traits, 2 independent lines of human PON3 transgenic (Tg) mice on the C57BL/6J (B6) background were constructed. Human PON3 mRNA was detected in various tissues, including liver, lung, kidney, brain, adipose, and aorta, of both lines of Tg mice. The human PON3 mRNA levels in the livers of PON3 Tg mice were 4- to 7-fold higher as compared with the endogenous mouse Pon3 mRNA levels. Human PON3 protein and activity were detected in the livers of Tg mice as well. No significant differences in plasma total, high-density lipoprotein, and very-low-density lipoprotein/low-density lipoprotein cholesterol and triglyceride and glucose levels were observed between the PON3 Tg and non-Tg mice. Interestingly, atherosclerotic lesion areas were significantly smaller in both lines of male PON3 Tg mice as compared with the male non-Tg littermates on B6 background fed an atherogenic diet. When bred onto the low-density lipoprotein receptor knockout mouse background, the male PON3 Tg mice also exhibited decreased atherosclerotic lesion areas and decreased expression of monocyte chemoattractant protein-1 in the aorta as compared with the male non-Tg littermates. In addition, decreased adiposity and lower circulating leptin levels were observed in both lines of male PON3 Tg mice as compared with the male non-Tg mice. In an F2 cross, adipose Pon3 mRNA levels inversely correlated with adiposity and related traits. Our study demonstrates that elevated PON3 expression significantly decreases atherosclerotic lesion formation and adiposity in male mice. PON3 may play an important role in protection against obesity and atherosclerosis.
atherosclerosis; obesity; genetics
Rationale and Objective
In this Emerging Science Review, we discuss a systems genetics strategy, which we call Gene Module Association Study (GMAS), as a novel approach complementing Genome Wide Association Studies (GWAS), to understand complex diseases by focusing on how genes work together in groups rather than singly.
The first step is to characterize phenotypic differences among a genetically diverse population. The second step is to use gene expression microarray (or other high throughput) data from the population to construct gene co-expression networks. Co-expression analysis typically groups 20,000 genes into 20–30 modules containing 10’s to 100’s of genes, whose aggregate behavior can be represented by the module’s “eigengene.” The third step is to correlate expression patterns with phenotype, as in GWAS, only applied to eigengenes instead of SNPs.
Results and Conclusions
The goal of the GMAS approach is to identify groups of co-regulated genes that explain complex traits from a systems perspective. From an evolutionary standpoint, we hypothesize that variability in eigengene patterns reflects the “good enough solution” concept, that biological systems are sufficiently complex so that many possible combinations of the same elements (in this case eigengenes) can produce an equivalent output, i.e. a “good enough solution” to accomplish normal biological functions. However, when faced with environmental stresses, some “good enough solutions” adapt better than others, explaining individual variability to disease and drug susceptibility. If validated, GMAS may imply that common polygenic diseases are related as much to group interactions between normal genes, as to multiple gene mutations.
systems genetics; genetics of complex diseases; scale-free networks; hybrid mouse diversity panel; computational biology
The protein phosphatase 1-like gene (PPM1l) was identified as causal gene for obesity and metabolic abnormalities in mice. However, the underlying mechanisms were unknown. In this report, we find PPM1l encodes an endoplasmic reticulum (ER) membrane targeted protein phosphatase (PP2Ce) and has specific activity to basal and ER stress induced auto-phosphorylation of Inositol-REquiring protein-1 (IRE1). PP2Ce inactivation resulted in elevated IRE1 phosphorylation and higher expression of XBP-1, CHOP, and BiP at basal. However, ER stress stimulated XBP-1 and BiP induction was blunted while CHOP induction was further enhanced in PP2Ce null cells. PP2Ce protein levels are significantly induced during adipogenesis in vitro and are necessary for normal adipocyte maturation. Finally, we provide evidence that common genetic variation of PPM11 gene is significantly associated with human lipid profile. Therefore, PPM1l mediated IRE1 regulation and downstream ER stress signaling is a plausible molecular basis for its role in metabolic regulation and disorder.
IRE1; PPM1l; Protein phosphatase; ER stress; Adipogenesis
To fine map previously identified quantitative trait loci (QTL) affecting atherosclerosis in mice using association analysis.
Methods and Results
We recently showed that high-resolution association analysis using common inbred strains of mice is feasible if corrected for population structure. To utilize this approach for atherosclerosis, which requires a sensitizing mutation, we bred human apoB100 transgenic mice with 22 different inbred strains to produce F1 heterozygotes. Mice carrying the dominant transgene were tested for association with high-density SNP maps. Here we focus on high-resolution mapping of the previously described Ath30 locus on Chr 1. As compared to the previous linkage analysis, association improved the resolution of the Ath30 locus by more than an order of magnitude. Using expression quantitative trait locus analysis, we identified one of the genes in the region, Des, as a strong candidate.
Our high-resolution mapping approach accurately identifies and fine maps known atherosclerosis QTL. These results suggest that high-resolution genome-wide association analysis for atherosclerosis is feasible in mice.
We investigated the association of glycemia and 43 genetic risk variants for hyperglycemia/type 2 diabetes with amino acid levels in the population-based Metabolic Syndrome in Men (METSIM) Study, including 9,369 nondiabetic or newly diagnosed type 2 diabetic Finnish men. Plasma levels of eight amino acids were measured with proton nuclear magnetic resonance spectroscopy. Increasing fasting and 2-h plasma glucose levels were associated with increasing levels of several amino acids and decreasing levels of histidine and glutamine. Alanine, leucine, isoleucine, tyrosine, and glutamine predicted incident type 2 diabetes in a 4.7-year follow-up of the METSIM Study, and their effects were largely mediated by insulin resistance (except for glutamine). We also found significant correlations between insulin sensitivity (Matsuda insulin sensitivity index) and mRNA expression of genes regulating amino acid degradation in 200 subcutaneous adipose tissue samples. Only 1 of 43 risk single nucleotide polymorphisms for type 2 diabetes or hyperglycemia, the glucose-increasing major C allele of rs780094 of GCKR, was significantly associated with decreased levels of alanine and isoleucine and elevated levels of glutamine. In conclusion, the levels of branched-chain, aromatic amino acids and alanine increased and the levels of glutamine and histidine decreased with increasing glycemia, reflecting, at least in part, insulin resistance. Only one single nucleotide polymorphism regulating hyperglycemia was significantly associated with amino acid levels.
The liver X receptor (LXR) signaling pathway is an important modulator of
atherosclerosis, but the relative importance of the two LXRs in atheroprotection is
incompletely understood. We show here that LXRα, the dominant LXR isotype
expressed in liver, plays a particularly important role in whole-body sterol
homeostasis. In the context of the ApoE−/− background,
deletion of LXRα, but not LXRβ, led to prominent increases in
atherosclerosis and peripheral cholesterol accumulation. However, combined loss of
LXRα and LXRβ on the ApoE−/− background led to an
even more severe cholesterol accumulation phenotype compared to
indicating that LXRβ does contribute to reverse cholesterol transport (RCT) but
that this contribution is quantitatively less important than that of LXRα.
Unexpectedly, macrophages did not appear to underlie the differential phenotype of
LXRβ−/−ApoE−/− mice, as in
vitro assays revealed no difference in the efficiency of cholesterol efflux from
isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled
macrophages revealed a marked defect in fecal sterol efflux in
Mechanistically, this defect was linked to a specific requirement for
LXRα−/− in the expression of hepatic LXR target genes
involved in sterol transport and metabolism. These studies reveal a previously
unrecognized requirement for hepatic LXRα for optimal reverse cholesterol
transport in mice.
atherosclerosis; nuclear receptor; cholesterol metabolism; apoliporotein
Coronary artery disease; genome-wide association; mouse atherosclerosis; myocardial infarction; plasma lipoproteins; systems genetics